Biochem Chp 21: Proton Motive Force

ETC

proton motive force is the

proton gradient generated by the oxidation of NADH and FADH2 which powers ATP synthesis

proton motive force ( delta p) =

chemical gradient (delta pH) + charge gradient (delta psi)

protons are pumped across the

inner mitochondrial membrane as electrons flow through the respiratory chain

the ETC is coupled to

ATP synthesis by the proton motive force: the ETC creates the PMF by pumping protons across the mitochondrial membrane and this gradient is then used by ATP synthase to synthesize ATP.

Also possible for mitochondria to experience uncoupling where the proton gradient is dissipated without driving ATP synthesis. Happens in brown adipose tissue where uncoupling proteins dissipate the proton gradient to generate heat instead of ATP.

chemiosmotic hypothesis

the electron transport and ATP synthesis are coupled by a proton gradient across the inner mitochondrial membrane (intact and impermeable to ptons); [H+] becomes lower in matrix and an E field with matrix side negative is generated → protons flow back into matrix to equalize the distribution and this flow drives the synthesis of ATP by ATP synthase

F1 component of ATP synthase contains

the active sites and protrudes into the mitochondrial matrix

ATP synthase bind to one another to form

dimers which then oligomerize. The oligomers contribute to cristae formation which creates an area where the photons have ready access to the ATP synthase

each ATP synthase has how many active sites located where

3 active sites located on the 3 beta subunits

the F0 component is embedded in the

inner mitochondrial membrane and contains the proton channel

the gamma subunits connects

the F1 and F0 components

each beta subunit is distinct in that each subunit

interacts differently with the gamma subunit

mechanism of ATP synthase

1. protons enter the F₀ from the intermembrane space or thylakoid lumen, where they are at a higher concentration due to the activity of the ETC

2. the flow of protons through the F₀ causes it to rotate. This rotation is transferred to the central stalk of F₁ unit

3. the rotational energy is then converted into chemical energy as the F₁ unit, which contains catalytic sites, facilitates the binding of ADP and inorganic phosphate and their condensation into ATP

4. the newly synthesized ATP is then released for the cell to use as an energy source

significance of dimerization of ATP synthase

1. membrane curvature

2. efficiency

3. structural organization

4. regulation of activity

proton flow occurs through the

F0 component of the ATP synthase: protons enter the half channel facing the pton rich intermembrane space, bind to a glutamate residue on one of the subunits of the c ring, and then leave the the c subunit when they rotate to face the matrix side of the channel

what powers the rotation of the c ring

the force of the proton gradient

the rotation of the c ring power what which does what

powers the movement of the gamma subunit which in turn alters the conformation of the beta subunits

the number of c rings determines the

number of protons required to synthesize a molecule of ATP

organisms with lower number of c subunits would be more efficient bc they require less protons to synthesize one ATP

the binding change mechanism

as the gamma subunit rotates it interacts differently with each beta subunit causing them to change conformation. This rotation leads to a sequential change in the conformation of each beta subunit cycling through the O,L, and T states. ensures as that as one beta subunit releases ATP (O state) another is synthesizing ATP (T state) and another is binding ADP and Pi (L state)

in the O (open) state,

nucleotides can bind to or be released from the beta subunit

beta subunit has a low affinity for nucleotides so ADP and Pi can enter and ATP can be released

in the L (loose) form, nucleotides

are trapped in the beta subunit

does NOT catalyze ATP synthesis

in the T (tight) form

ATP is synthesized from ADP + P_i

actual synthesis of ATP occurs.

T/F: no two subunits are every in the same conformation

true

the rotation of the gamma subunit interconverts

the beta subunits

oxidative phosphorylation refers to how the

ETC generates a proton gradient which is then used to synthesize ATP

why do isolated F₁ subunits of ATP synthase catalyze ATP hydrolysis?

the hydrolysis of ATP is exergonic so ATP synthase will enhance the hydrolytic reaction

ATP synthases isolated from different sources often have different numbers of c subunits. what effect would altering the number of c subunits have on the yield of ATP as a function of proton flow?

the number of c subunits determines the number of protons that must be transported to generate a molecule of ATP. ATP synthase must rotate 360 degrees to synthesize 3 molecules of ATP, so the more c subunits there are, the more protons are required to rotate the F1 units 360 degrees

in muscle (or tissues were rapid ATP generation is critical) electrons from cytoplasmic NADH can enter the ETC by using the

glycerol phosphate shuttle

in the glycerol phosphate shuttle the electrons are transfered from

NADH to FADH₂ and subsequently to Q to form QH₂

why is glycerol-3-phosphate shuttle less efficient than malate aspartate shuttle?

it results in the production of FADH₂ in the mitochondria which contributes fewer ATPs per molecule oxidized compared to NADH

which shuttle is faster

glycerol-3-phosphate shuttle is faster, allowing for a quicker regeneration of NAD+ in the cytosol which is essential for continuation of glycolysis during highE demands

where is the malate aspartate shuttle used?

used in tissues where the efficiency of ATP production is more important (liver, heart, kidney)

why is the malate aspartate shuttle more efficient?

it directly transfers the reducing equivalents into the mitochondria as NADH which then enters the ETC at a point that yields more ATP; it is slower but MAXIMIZES the ATP yield from glucose oxidation (more efficient)

malate aspartate shuttle uses the

electrons from cytoplasmic NADH to generate mitochondrial NADH; consists of 2 membrane transporters and 4 enzymes

2 main transporters in malate aspartate shuttle

malate alpha ketoglutarate transporter (malate shuttle): antiporter that exchanges malate from the cytosol with alpha-ketoglutarate from the mitochondria facilitating entry and exit of malate and alpha-ketoglutarate across the inner mitochondrial membrane

glutamate-aspartate transporter: moves aspartate out of the mitochondria in exchange for glutamate coming into the mitochondria. the aspartate can then be converted back to OAA in the cytosol

what enables the exchange of cytoplasmic ADP for mitochondrial ATP

ATP-ADP translocase: one molecule of ADP from the cytosol must enter the mitochondria for one molecule of ATP to leave from the mitochondrial matrix

ATP-ADP translocase is powered by

proton motive force

what 3 components come together to form the ATP synthasome

ATP-ADP translocase, the phosphate carrier, and ATP synthase

the complete oxidation of glucose yields about how many molecules of ATP?

of the total number, how many are formed in ox phos?

how are the remaining formed?

30 ATP are formed by the complete oxidation of glucose

26 are formed in oxidative phosphorylation

4 are yielded by the metabolism of glucose to 2 molecules of pyruvate in glycolysis

when glucose undergoes fermentation how many molecules of ATP are generated per glucose?

2 molecules of ATP are generated per glucose

the rate of oxidative phosphorylation is determined by

the need for ATP; electrons do not flow through the ETC unless ADP is available to be converted into ATP

acceptor or respiratory control refers to

the regulation of oxidative phosphorylation by ADP

acceptor control is control of metabolism by

energy charge

energy charge acts as a cellular “fuel gauge” that helps regulate the ETC and ATP synthesis through 3 mechanisms

1. direct ATP synthase regulation: high ATP levels (high E charge), ATP itself acts as allosteric inhibitor. high ATP levels slow down ATP synthase activity

2. upstream control: higher energy charge reduces electron flow through NADH and FADH2 pathways. CAC also inhibited, reducing electron input to the system

3. proton gradient regulation: proton gradient is maintained at an optimal level. if ATP levels are high → proton pumping slows down; if ATP levels are low (low E charge) → proton pumping increases to drive more ATP synthesis

regulatory system ensures that cells don’t waste energy making excess ATP when stores are full, ATP prod can quickly ramp up when E charge drops, and proton gradient is maintained at efficient levels

what does inhibitory factor 1 do?

inhibits ATP synthase and may prevent ATP hydrolysis when oxygen is limited

inhibitory factor 1 is overexpressed in some cancers and may

facilitate the transition to aerobic glycolysis

nonshivering thermogenesis is when

electron transport is uncoupled from ATP synthesis and heat is generated; facilitated in a regulated fashion by uncoupling protein 1 (UCP1) aka thermogenin (integral protein of inner mitochondrial membrane)

where does uncoupling occur

in mitochondria in brown fat aka brown fat mitochondria; adults display nonshivering thermogenesis

inhibition of the ETC prevents ox phos by

inhibiting the formation of the proton motive force

inhibition of ATP synthase by inhibiting proton flow prevents

electron transport

uncouplers carry protons across the inner mitochondrial membrane. what still functions and why is ox phos still inhibited

the ETC still functions but ATP synthesis doesn’t occur because the proton gradient can never form

inhibition of the ATP-ADP translocase

prevents ox phos

disruption in which complex is the most common cause of mitochondrial disease

complex 1; defects in ETC components reduces ATP synthesis and also increases the amount of ROS formed → increased mitochondrial damage